In collaboration with clinical investigators in the NIH IRP, we are continuing our analysis of the antiviral immune response of patients individuals who have developed acute and chronic HCV infection. The aim of these studies is to compare CD4 and CD8 T cell responses in persons whose infection resolves either spontaneously or after interferon-based treatment to those who are persistently infected, to determine factors contribute to the exhaustion of HCV-specific T cell responses in persistent infection and to identify the mechanisms responsible for defective immune responses in chronic infection.[unreadable] [unreadable] In collaboration with extramural investigators, we have completed and published the analysis of immune responses of injection drug users (IDUs) who have demonstrated a reduced risk of developing chronic re-infection despite continuing exposure to the virus. IDUs constitute the largest group of HCV-infected people in the United States and account for 42% of new infections. After 10 years of injection drug use, 90% test anti-HCV positive in a standard enzyme immunoassay, of whom 80% are persistently HCV-infected (Lorvick et al., Am J Public Health 2001; 91:46-7). However, two recent epidemiological studies demonstrated that IDUs who successfully cleared HCV in the past have a reduced risk of developing persistent HCV viremia despite continued injection drug use and continued exposure to HCV (Mehta et al., Lancet 2002; 359:1478-83; Grebely et al., Hepatology 2006; 44:1139-45). Subjects who had cleared a past HCV infection were 2-times (Mehta et al., Lancet 2002; 359:1478-83) and 4-times (Grebely et al., Hepatology 2006; 44:1139-45) less likely to develop persistent HCV infection than subjects without evidence of past HCV exposure. To identify potential immunological correlates of immune protection we studied cellular and humoral immune responses of a subgroup of IDUs who reported more than 10 years of injection drug use. [unreadable] [unreadable] Studying long-term (>10 years) IDUs, we observed that nonviremic EIA-positive IDUs displayed more frequent and stronger HCV-specific T-cell responses than viremic EIA-positive IDUs. This correlation held true for both proliferation and IFN-&#947; production of HCV-specific T-cells and was observed in response to both recombinant HCV proteins and overlapping peptides. The greatest difference in response strength between the groups was observed to HCV core and NS3. The frequency and strength of the T-cell responses of these frequently re-exposed IDUs was comparable to those previously observed in subjects who have been exposed only once in their lifetime (Takaki et al., Nature Medicine 2000; 6:578-82; Lechner et al., J Exp Med 2000; 191:1499-512)[unreadable] [unreadable] In contrast to HCV-specific T-cell responses, the frequency of subjects with neutralizing antibodies (nAb) was greater in viremic EIA-positive than in nonviremic EIA-positive IDUs. The high frequency of nAb is consistent with the literature, which describes these responses developing late in infection. However, nAb appear ineffective at clearing autologous virus at the time of sampling due to the emergence of viral escape variant (von Hahn, Gastroenterology 2007; 132:667-78). Overall nAb were detected in more individuals than expected from the literature (Bartosch, Proc Natl Acad Sci U S A 2003; 100:14199-204). These results may be attributable to the fact that the nonviremic EIA-positive IDUs have likely been frequently re-exposed to HCV due to continuing IDU, whereas published cohorts consist predominantly of individuals who were singly exposed to the virus. Indeed, prospective studies demonstrate that HCV-specific antibodies decline and can become undetectable after spontaneous (Takaki et al., Nature Medicine 2000; 6:578-82) and therapy-induced clearance of HCV (Wiegand et al., Hepatology 2004; 40:98-107). These findings suggest that continued, long-term exposure to HCV antigens is required to maintain humoral immune response. [unreadable] [unreadable] HCV-exposed, nonviremic EIA-negative IDUs frequently mounted T-cell responses with detectable proliferation, IFN-g production and cytotoxicity against structural and nonstructural HCV antigens. These subjects may have either lost all HCV-specific antibodies after clearance of a previous HCV infection or may have never mounted antibody responses. The presence of CD8+ T cells that target HCV nonstructural sequences is evidence that HCV RNA translation and/or replication must have occurred at least briefly because CD8 T cell responses against epitopes in nonstructural proteins are either directly primed by virus-infected cells or crossprimed by cells that have engulfed virus-infected cells. This detection of HCV-specific T-cell responses in nonviremic EIA-negative IDUs differs from a previous study, which did not detect T-cell responses in 8 IDUs who remained HCV EIA-negative for 1 year of follow-up, despite IDU patterns associated with a high risk for HCV transmission (Wong et al., J Virol 2001; 75:1229-35). The different results may be explained by differences in study populations and immunological techniques. The published report analyzed subjects had recently started injecting drugs (Wong et al., J Virol 2001; 75:1229-35), and their cumulative HCV exposure was therefore lower than that of the long-term (>10 years) IDUs in our study. Moreover, the published report was based on an in vitro T-cell expansion technique with vaccinia virus-infected antigen-presenting cells (Wong et al., J Virol 2001; 75:1229-35), which is less sensitive than the ex vivo IFN-&#947; ELISpot technique with overlapping HCV peptides used in our study. [unreadable] [unreadable] Our results are consistent with the detection of HCV-specific T-cell responses by IFN-&#947; ELISpot analysis in nonviremic EIA-negative family members of HCV-viremic patients (Koziel et al., J. Infect. Dis. 1997; 176:859-66; Bronowicki et al., J. Infect. Dis. 1997; 176:518-22) and are also reminiscent of HIV-specific T-cell responses in prostitutes who remained HIV-negative despite continued high-risk exposure (Rowland-Jones et al., J. Clin. Invest. 1998; 102:1758-65). In that report, a break from sex work for about a year was associated with a loss of HIV-specific CD8+ T-cell responses and a higher risk of HIV viremia and seroconversion (Kaul et al., J Clin Invest 2001; 107:341-9). These results, along with the epidemiological finding that HCV-nonviremic EIA-positive IDUs have a reduced risk of acquiring persistent infection Mehta et al., Lancet 2002; 359:1478-83; Grebely et al., Hepatology 2006; 44:1139-45), suggest T-cell-mediated immunity. Thus, HCV-specific T-cell responses may contribute to the apparent immune protection in this cohort of longterm (>10 years) IDUs.